Tissue-type plasminogen activator is the only therapy for the acute thromboembolic stroke, which is approved by the Food and Drug Administration (FDA). However, there is reason for concern that use of tPA for treatment of ischemic stroke may expose patients to secondary intracerebral hemorrhage. Wardlaw J C et al, Lancet 1997, 350:607–614. This is because there is an approximately six percent incidence of subsequent symptomatic intracerebral hemorrhage and approximately fifty percent of these patients die. The appearance of intracerebral hemorrhage after treatment with tPA is attributed to its capacity to interfere with the normal vasoactivity of the cerebral blood vessels. TPA has been shown to have dose-dependent vasoconstrictory or vasodilatory effects besides promoting the activation of plasminogen.
Tissue-type plasminogen activator is a naturally occurring molecule released from vascular endothelial cells, and rapid removal of tPA from the blood occurs by clearance in the liver. Hepatocytes express the low-density lipoprotein receptor-related protein or d2-macroglobulin receptor which binds tPA and complexes of plasminogen activator inhibitor (PAI-1) with tPA and tcuPA. Alternately, endothelial cells express a 170 Kda mannose-dependent receptor which is also involved in the rapid clearance of tPA.
Pro-uroldnase (Pro-UK) also known as single chain urokinase plasminogen activator (scuPA), is a naturally occurring molecule released from vascular endothelial cells in response to formation of blood clots and other pathological conditions. ScuPA or Pro-UK can be activated by two different mechanisms a) by cleavage of a single peptide bond by plasmin that leads to the generation of the active form composed of two chains (tcuPA) and b) by binding of scuPA to its receptor, urokinase plasminogen activator receptor (uPAR).
Plasminogen activator inhibitor type 1 (PAI-1) binds to tcuPA and inhibits its catalytic activity. However, PAI-1, which binds tcuPA with high affinity, binds with only low affinity, if at all, to scuPA.
Plasminogen activator inhibitor type 1 interacts with both tPA and uPA and inhibits the catalytic activity of both proteins. PAI-1, which binds tPA and uPA with high affinity is present at high concentrations in the circulation of patients suffering from hypertension. And, reduction of blood pressure by medical treatment results in a decrease of PAI-1 concentrations. The underlying mechanism of action for the increase of PAI-1 in certain pathological conditions is not understood well. However, the inverse relationship with tPA and/or uPA suggests that PAI-1 serves to neutralize in some way the vasoactive effect of tPA and/or uPA. Simmons M, Cardiol. Clin 1995, 13:339–345; Cipolla M et al., Stroke, 2000, 31:940–945; of PAI-1; and Higazi, A. A.-R et al., J. Biol. Chem., 1995, 270:9472–9477.
The question of whether there is a link between increased levels of PAI-1 concentrations in certain pathological conditions and naturally produced tPA, or whether there is a link between PAI-1 and intracerebral hemorrhage due to use of commercially produced tPA, has not been evaluated heretofore. The present invention is directed to gain a better understanding of the control if any, of PAI-1 or tPA or uPA, and to providing a composition or product optimally effective at regulating activity of tPA or uPA, thereby reducing the risk of intracerebral hemorrhage in patients receiving thrombolytic therapy such as tPA and/or uPA.
Several approaches to thrombolytic therapy have been under investigation, one being through the systemic infusion of activators of the naturally occurring or commercially produced recombinant varieties of fibrinolytic agents. Urokinase is a thrombolytic agent active through the conversion of plasminogen to plasmin. Urokinase is a complex protein of unknown structure which is found in urine in trace amounts. Recombinant forms of urokinase have been developed and are being tested for clinical efficacy, for example U.S. Pat. No. 4,558,010 issued to Abbott Laboratories, describes a recombinant deoxyribonucleic acid which codes for the plasminogen activator protein having human urokinase activity.
The present invention demonstrates that a six amino acid peptide (SEQ ID NO:1) or an eighteen amino acid peptide (SEQ ID NO:2), can reduce the undesirable side effects of fibrinolytic agents, for example, the risk of intracerebral hemorrhage in patients receiving tPA, uPA, tcuPA, streptokinase, rt-PA or alteplase, rt-PA derivatives or anisoylated streptokinase complex. In the protocol employed, the peptide was introduced into the thrombolytic regimen in later stages to prevent the vasoactive or side effects of the primary thrombolytic agent.
The question of whether the peptide has any effect when administered in the early stage of the thrombolytic therapy, has not been investigated heretofore. The present invention describes some unexpected results obtained when the peptide is administered when combined with a plasminogen activator right from the start of the thrombolytic therapy. The results are unexpected because they demonstrate a synergistic effect when the peptide and the plasminogen activator are administered together in in vitro and in in vivo systems. The present invention thus provides novel compositions of different plasminogen activators and the peptide and methods for optimizing the efficacy of thrombolytic agents in combination therapeutic regimens. Such an approach suggests that the effective dosage of the thrombolytic agent can be reduced in the presence of the peptide. This in turn reduces the risk for side effects of these agents, the side effects being manifested in the late stage of therapy.